New Analytical RP-HPLC Method Development and Validation for the Estimation of Prucalopride in Bulk and Pharmaceutical Dosage Form

Introduction

Prucalopride is a licensed drug used for the treatment in women for chronic constipation. Chronic Idiopathic Constipation (CIC) is one of the most common chronic functional gastrointestinal disorder for which Prucalopride is used in the treatment.^1,2

Prucalopride is a first class dihydrobenzo furan carboxamide derivative which acts as a selective serotonin receptor agonist.³ This action results in increased release of acetylcholine in GIT (gastrointestinal tract), which is used in restoring bowel function by stimulating mobility in colon. Literature survey revealed few UV spectroscopic methods^4,5 and one High Performance Liquid Chromatography (HPLC) method⁶ for the drug Prucalopride. The present study was concentrated on RP-HPLC method mainly to decrease retention time than reported method in literature survey.

Materials and Methods

Apparatus and Software

The separation was carried out in RP-HPLC by Agilent 1120 compact LC system with column (Water’s X Bridge 5 µ C18(2) 100A, 250 X 4.6 mm), Mixer (Rhenodyne injector with 20 μl fixed loop, UV detector SPD 10 A yp), Gradient pump (LC- 10AT VP pump, 4 MPa or 40 bar), Agilent syringe, Vaccum pump (SUPER FIT 110336), Millipore nylon for sample and solvents filtration, Sonicator (EQITRO N 230 VAC, 50Hz) Analytical weighing balance (Shimadzu AU X220), and software (EZ- chrome elite software- double channel) was used for acquisition, evaluation and storage of chromatographic data. Double beam UV- visible spectrophotometer (SHIMADZU UV 1700) was used for wavelength detection.

Drug Sample

Commercially available Prucalopride 2 mg tablet were procured from local pharmacy shop, manufactured by Torrent Pharmaceuticals Limited, Gangtok, Sikkim. Pure drug was obtained as a gift sample from Torrent Pharmaceuticals Limited.

Preparation of mobile phase

For preparing mobile phase, HPLC grade methanol and orthophosphoric acid were used. Direct-Q water purification system (Millipore, Milford, USA) was used for preparing HPLC water.

Chromatography

For finalizing the method parameters, several trials with different ratio and different combinations of solvents were tried. The mobile phase 0.1% orthophosphoric acid: methanol (30:70 %v/v) showed reasonably better response and maximum peak at 225 nm.

Mobile Phase preparation

Mobile phase was prepared by using methanol and orthophosphoric acid. Seventy percent of methanol and 0.1% orthophosphoric acid (30%) were allowed to degas separately in ultrasonic water bath for 15 mins and filtered with 0.45 μ filter under vacuum filtration.

Linearity

The calibration curve with six concentrations of the standard drug solution 5-30 μg/mL was plotted and each dilution was repeated six times by taking area in Y axis and concentration in X axis. The linearity was evaluated by linear regression analysis and shown in figure 1.

Specificity and selectivity

To validate the parameter specificity and selectivity, the tablet formulation excipients were spiked and the absorbance was measured and calculated to determine the quantity of the drugs. Blank Chromatogram is shown in figure 2.

Precision

Precision for the method was studied by injecting the sample multiple times. Precision was done by repeatability (also called intermediate precision) and reproducibility methods. In these methods, precision was measured by using peak area and peak symmetry parameters of HPLC method. Six injections were made within and between the days and the obtained results of trials were in acceptable range. The results are shown in figure 3, 4, 5 and 6.

Accuracy

Accuracy validation was done with three different concentrations of drug. Prucalopride drug in concentrations of 80%, 100% & 120% were prepared in three 25 mL volumetric flasks separately. Areas of three concentrations were measured at 225 nm as shown in figure 7, 8 and 9.

Robustness

The robustness describes its capability to remain unaffected by deliberate and small changes in the chromatographic conditions as per International Conference of Harmonization (ICH) guidelines. It was found unaffected by small variation of ±0.1 mL/min in flow rate of mobile phase and ±1 nm in wavelength.

Limit of detection (LOD) and limit of quantification (LOQ)

As per ICH, the LOD and LOQ were calculated based on the signal-to-noise ratio 3:1 and 10:1, respectively.

Results

The present method was validated for all parameters as per International Conference of Harmonization (ICH) guidelines. The structure of Prucalopride is given in figure 10 and the results of all parameters are given in tables 1, 2, 3, 4 and 5.

Discussion

The Prucalopride drug estimation by HPLC was reported previously as per literature survey. The present study by using HPLC was mainly concentrated in decreasing retention time and succeeded in developing a method which got results for different validation parameters within the limits as per ICH.

Mobile phase 0.1% orthophosphoric acid: methanol (30:70 %v/v) was used at 225 nm UV detection with 15 min run time separated drug at retention time of 1.55 min.

Conclusion

The proposed RP-HPLC method was suitable technique for the determination of Prucalopride. The purpose of study was fulfilled by decreasing retention time to 1.5 minutes. All the parameters for analysing Prucalopride met the criteria as per ICH guidelines for method validation. By this investigation, we have developed a simple, sensitive, precise and accurate RP-HPLC method for the quantitative estimation of Prucalopride in bulk and pharmaceutical formulations. The HPLC method developed is recommended for the routine determination of Prucalopride.